RNA molecules display a novel mode of action in pond-dwelling ciliated protists, where both long and small noncoding RNAs regulate an intricate process of genome remodeling. Maternally-inherited piRNA molecules in Oxytricha mark which 225,000 segments of the genome to retain, and long, template RNAs program the order of those DNA segments during massive genome rearrangements. The proposed goal of this research is to understand the biogenesis and regulation of both these long and small noncoding RNAs, as well as the regulation of other steps in pathways of RNA production in the model organism Oxytricha. Expanding our suite of tools for studying and manipulating this emerging genetic system is also a high priority, and one aim will capitalize on our knowledge of piRNA biology in these organisms to develop a powerful tool for gene knockout. This will, in turn, help us better understand other aspects of the genetics and developmental biology of genome rearrangements, as well as its implications for the maintenance and regulation of genome integrity, more generally. The proposed experiments will examine the properties of both the molecules that participate in these complex rearrangement events and the machinery underlying them. The most immediate insight to be gained from the proposed research is an understanding of the range of activities accessible to both long and small noncoding RNA molecules, and the piRNA pathway in Oxytricha, in particular, with attention to both differences and similarities between this system and other eukaryotic organisms. Specific aims include: 1. Studying piRNAs and their biogenesis in Oxytricha, 2. Develop piRNA-mediated gene knockout as the first gene knockout tool in Oxytricha, 3. Understand long, noncoding template RNA production and their interacting partners, 4. Identify and functionally test motifs that regulate gene expression from somatic chromosomes.